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Capillary Electrophoresis Rachel K. Harstad, Alexander C. Johnson, Megan M. Weisenberger, and Michael T. Bowser Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04125 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015
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Analytical Chemistry
Capillary Electrophoresis Rachel K. Harstad, Alexander C. Johnson, Megan M. Weisenberger, Michael T. Bowser University of Minnesota, Department of Chemistry, 207 Pleasant Street South East, Minneapolis, MN 55455 Capillary electrophoresis (CE) is a field that continues to grow. All areas of CE including theory, separation modes, instrumentation, and applications remain active areas of research. This review includes a cross section of references from all areas of the field published in the 4 year period between January 2012 and September 2015. Web of Science reports over 7500 articles, including 663 reviews, published with CE in the title, abstract, or keywords during this time period. Of these we have chosen 216 papers. We have attempted to choose papers that showcase some of the newest and most exciting developments in the field. It should be noted that papers describing electrophoresis in microfabricated devices were excluded since another review in this issue exclusively covers this topic.
Techniques Fundamentals and Separation Modes. CE has emerged as a versatile and robust separation technique. As the technique has matured the number of publications that primarily focus on CE fundamentals and separation modes has declined. This downward trend is unfortunate as there is still much to be learned about CE and further innovation is needed to improve the technique for the greater scientific community. Pressure driven sample injections in CE can result in asymmetric peaks due to the parabolic flow profile caused by pressure driven flow. Kanoatov et al. describe a method for correcting this peak asymmetry.1 After the sample vial is replaced with running buffer, an additional pressure injection is performed to create a parabolic flow profile on both the front and back end of the sample plug. This corrects the pressure driven sample plug peak shape to be more symmetric. Amini described using double injection CE (DICE) as a means to account for poor migration time repeatability for peak identification.2 In DICE, the analytes are first injected on the capillary while a standard reference material is introduced in a second sample injection. Identification of analytes based on their migration times was demonstrated using a mixture of several pharmaceuticals. Li et al. reported using a new mode of CE for high-resolution separations of chiral compounds.3 This new technique is called velocity gap (VG) mode CE. In VG, two consecutive electric fields are applied to drive analytes through two connected capillaries with conductivity detection at the joint. By monitoring the velocity changes of the analytes as they travel through the two capillaries, the net velocity change for each analyte can be analyzed. When compared to traditional capillary zone electrophoresis (CZE), this new mode was able to achieve better resolution in separation of enantiomeric pairs. Huge et al. report new cost-effective fraction collection device for CE. This fraction collection device does not rely on prior knowledge of migration times of the analytes, or stoppage of the separation.4 This
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is accomplished with a CE-Matrix Assisted Laser Desorption/Ionization (CE-MALDI) interface design that uses an ink jet printer valve. Fractions are collected in a 96 well microtiter plate. The fraction collector was tested with CE-Systematic evolution of ligands by exponential enrichment (CE-SELEX) to test for thrombin binding aptamers. Zeng et al. report highly repeatable and accurate injection control at the picoliter level.5 The capillary is raised out of the running buffer well to receive a droplet from a modified ink jet printer microchip. Once the sample plug was loaded, the capillary is automatically submerged back into the running buffer. The peak area % relative standard deviation (RSD) for four injections of taurine in a urine sample was 1.05%. Analytes were separated using CZE and were electrophoretically transferred into droplets using an interface developed by Keyon et al.6 The end of the separation capillary is connected to a cross where droplets are flowing perpendicularly. Analytes are then pushed into the droplets where they flow to the detection apparatus. Paralytic shellfish toxins were separated in the separation capillary and then transferred to the droplets containing labeling reagents. The toxins were then detected with laser induced fluorescence. Compartmentalizing the toxins reduces degradation of the analytes and allows for preservation of CZE separation while gaining sensitivity for fluorescence detection.
Sampling and Preconcentration. Studies presented below employ techniques and protocols designed to increase sampling efficiency. Decreasing sampling time and optimizing sampling protocols were of high importance in the immediate section, with subsections below further highlighting various sampling techniques such as affinity-based, electrophoretic, and pH mediated sampling. Sampling The ratio of sample injected to the total sample amount required for injection is known as injection efficiency, with typical values ranging from 10 to 10. Grundmann et al. designed a capillary batch injection, compatible with both nonaqueous and aqueous background electrolytes, capable of injecting extremely small samples (nL range) very efficiently.7 Injection efficiencies up to 80% were able to be achieved while maintaining the separation via CE-Mass Spectrometry (MS). To study the interaction of human serum albumin (HSA) with trimetazidine dihydrochloride (TMZ), Sun et al. built an ultrasonic microdialysis device to couple with CE-electrochemiluminescence (ECL).8 Traditional dialysis devices require 240 minutes of equilibration time, while the ultrasonic microdialysis device constructed requires only 45 minutes. The improved efficiency of the experimental procedure with a shortened experiment time yielded a limit of detection (LOD) of 26 nmol/L. Subcritical water extraction (SWE) was compared with chloroform soaking extraction, water ultrasonic extraction and accelerated solvent extraction (ASE) for five alkaloids by Wang et al.9 SWE was found to have the most abundant total alkaloid yields and with water as a solvent, it can be directly injected onto CE for analysis. SWE- field amplified sample stacking (FASS)-CE produced LODs of 0.004-0.0013 µg/mL.
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Wang et al. developed a method that decreased preparation time and solvent use by ultrasoundassisted emulsification microextraction and solidification of floating organic droplet (UAEM-SFO). Sample and extraction solutions were placed in an ultrasonic bath, and upon sonification, emulsification became suspended throughout the sample. The sample was then centrifuged and cooled in an ice bath, where the organic solvent was solidified and transferred to another vial to be quickly melted. Extractant was then redissolved in solution for introduction onto high performance CE. The entire procedure was optimized to require only 40 µL of extraction solvent and total pretreatment time was 8 minutes.10 Affinity-Based Preconcentration. Studies using affinity-based sampling techniques are examined in this subsection with their ability to increase the efficiency of sampling protocols and to increase enhancement factors. There was a heavy focus on optimizing current solid phase extraction (SPE) techniques, through the use of mixed cation exchange, varying the sorbent used and the integration of monolithic molecularly imprinted polymer (MIP) fibers. Xue et al. coupled dispersive liquid-liquid microextraction (DLLME ) with High Performance (HP)-CZE for the extraction of preservatives in cosmetic products.11 Small solvent consumption makes the protocol more environmentally benign, with LODs ranging from 0.200 to 0.375 mg/kg. A novel extraction protocol was developed by Hernández-Mesa et al. using a SPE cartridge with a mixed cation exchange for the analysis of 5-nitroimidazoles in milk.12 Sweeping effects were achieved for all studied compounds, an off-line concentration factor of 18 was recorded and LODs for all studied compounds were 500 ng/mL total DNA. Zhang et al. used a combination CE and HPLC-MS/MS to screen inhibitors of rapamycin (mTOR). CE yielded quantitative information regarding enzyme inhibition and inhibition kinetics while HPLC-MS/MS provided structural characterization.190 Through this technique, salvianolic acid A and C in Salvia miltiorrhiza extracts were found to be new inhibitors of mTOR. Resistance to β-lactum antibiotics is primarily due to the presence of β-lactamases. To provide a technique for organism identification, Fleurbaaij et al. developed a novel CE-ESI-MS/MS bottom-up proteomics approach for the analysis of peptides.191 This method was validated for its detection and identification of OXA-48 and KPC β-lactamases, in various species, even when present in single bacterial colonies. Microdialysis sampling as an in vitro technique has previously been limited by long sampling times and 10-20 minute temporal resolutions due to dilution of species in bulk solution. Hogerton and Bowser developed a novel in vitro technique coupling microdialysis sampling to CE-LIF.192 Immortalized astrocyte cells (C8-D1A) were grown directly on the probe membrane, allowing for fast sampling times with 20 second temporal resolution. Significant changes in amino acid analyte release, including glycine and Dserine, were detected when cells were exposed to a high potassium solution.
Environmental. MS was the common detection technique coupled with CE for the analysis of various environmental samples. Analytes examined ranged from polycyclic aromatic hydrocarbons (PAHs) to chloroanilines, with a heavy emphasis being placed on herbicides and water sample testing.
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Liu et al. used a CE-ESI-MS sprayer kit to effectively interface CE with inductively coupled plasma (ICP)-MS for the simultaneous analysis of ten arsenic compounds.193 Separations were achieved within 30 minutes with LODs ranging from 0.9-3.0 ng As/g. Herbal plant, chicken meat, ground water and two reference materials (TORT-2 and DORM-3) were successfully analyzed (see Figure 8). In order to provide a safe method for analyzing MeHg, EtHg and Hg (II) simultaneously, Zhao et al. coupled an Figure 8 Arsenic detected in environmental samples by environmentally-friendly microwave-assisted CE-ICP-MS. Reprinted from Journal of Chromatography extraction technique with CE-ICP-MS.194 A, Vol 1304, Liu, L.; He, B.; Yun, Z.; Sun, J.; Jiang, G., Detection limits were 0.021, 0.027 and 0.032 Speciation analysis of arsenic compounds by capillary electrophoresis on-line coupled with inductively ngHg/mL for MeHg, Hg (II) and EtHg, coupled plasma mass spectrometry using a novel respectively. Analytes were examined in both interface, pp 227-233 (ref 193). Copyright 2013 with natural water and fish muscle, with dried fish permission from Elsevier B.V. muscle showing only MeHg present. River water samples had undetectable levels of mercury, indicating they fall below the detection limits of the system. Pan et al. developed a method for the analysis of trace chloroanilines in water samples using hollow fiber-based liquid-phase microextraction with CE and amperometric detection.195 No derivatization was required for this assay, while still providing recoveries over 80% for sewage and environmental water samples. Eight aniline compounds were separated in 25 minutes with LODs ranging 0.01-0.1 ng/L. Phenoxy acid herbicides can contaminate environmental waters and reach toxic levels due to their high water solubility. Tabani et al. developed a low voltage EME method with CD modified CE for the determination and quantification of phenoxy acid herbicides.196 LODs were determined to be 10-15 ng/mL with LOQs of 30-40 ng/mL for three common herbicides in river water samples. Information regarding the molecular structure of organic compounds present in aerosols is critical for the development of assays to determine their effect on climate change. Yassine et al. were able to identify over 100 acidic components of organic aerosols using CE-MS and off-line Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS).197 These acidic components included aliphatic and aromatic carboxylic acids, nitrogen-containing carboxylic acids, organosulfates, and (nitrooxy)organosulfates. PAHs are some of the most targeted pollutants by international regulatory agencies. Ferey et al. developed a CE-LIF method for the rapid analysis of PAHs using a combination of methyl-β-cyclodextrin
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and sulfobutyl-β-cyclodextrin.198 All eight heavy PAHs of interest were baseline resolved in less than 7 minutes. Hu et al. used CE-ECL for the simultaneous detection of three phenylurea herbicides.199 With the optimized method, LODs for monuron, monolinuron and diuron were 0.05, 0.04 and 0.01 µg/L, respectively. Environmental contamination by herbicides containing triazines can be hazardous to human health. Fang et al. developed an on-line sweeping MEKC method to examine eight triazine herbicides in vegetable and cereal samples.200 The on-line sweeping technique provided preconcentration without requiring purification procedures typically needed for such a complex matrix. LOQs were determined to be 0.1 ng/g for all eight triazines. Warren et al. developed a CE-MS/MS assay to assess the composition of small peptides in soil samples.201 According to previous work, the soil sample was assumed to contain at least 298 peptides. It was discovered that less than 5% of peptides studied contained basic amino acid residues, indicating acidic and neutral amino acids were preferably retained in the soil. Microbial communities are typically studied using polymer 4 (POP-4), which separates based on amplicon length. Damaso et al. employed the use of F-108 polymer with CE on four bacteria species whom all produce the same length amplicon in the 16S rRNA V3 domain, but each with different nucleotide sequences.202 F-108 polymer yielded 4 separate peaks for the four species tested while POP-4 showed one peak. An environmental sample of hot spring microbial mat displayed 15 peaks using F-108 polymer while POP-4 only yielded 6. Bioprocessing of pine bark through the use of fungi can lead to organic acids and inorganic anions present in the extract. Boke et al. identified nine organic acids simultaneously in fermented samples within 12 minutes. Three inorganic anions were detected in less than 10 minutes, with the inorganic anions detected concurrently within the same separation. 203 Estrogens are pollutants to the environment which can contaminate water through their presence in animal-based fertilizers. D’Orazio et al. used DLLME-MEKC-MS to analyze 12 estrogenic compounds in mineral, run-off and waste water samples.204 All 12 analytes were able to be detected using ammonium perfluorooctanoate (APFO) as the volatile surfactant, with LODs of 0.04-1.10 µg/L.
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Figure 9. Network of plant metabolite correlations as obtained based on Pearson correlation coefficient values above the p>0.65 threshold. With kind permission from Springer+Business Media: Meabolomics, Study of polar metabolites in tobacco from different geographical origins by using capillary electrophoresis–mass spectrometry, Vol 10, 2014, pp 805-815, Zhao, J.; Hu, C.; Zeng, J.; Zhao, Y.; Zhang, J.; Chang, Y.; Li, L.; Zhao, C.; Lu, X.; Xu, G, (ref. 205) Figure 4. Copyright 2014 with permission of Springer. The study of plant metabolomics can used to understand their responses to changing environmentaconditions. Zhao et al. studied polar metabolites in tobacco leaves from two different geographical locations using CE-TOF-MS.205 A total of 154 metabolites were able to be identified (see Figure 9). Key photosynthesis metabolites (glycine, GABA, serine, and others) demonstrated changes in response to environmental conditions.
Materials. A significant portion of studies examined demonstrated the analysis of nanoparticles, with focus ranging from their migration behavior, to their effective charges and to their content in dietary supplements. Movement of nanoparticles was also able to be tracked for the first time in real time. Other natural compounds such as cellulose, lignocellulose, and rosin were also studied. Causon et al. used CE-quadrupole-time-of-flight (Q/TOF)-MS to identify the chemically decomposed components of four polyimides.206 Three different carboxylic acids were determined to be present. The diamine components, which are typically used to distinguish among various polyimide structures, yielded
